This analysis is to look at the MDS samples that were treated with the drug HTT homoharringtonine which is a translation inhibitor. There were only 2 samples for each treatment in this arm.
library(dplyr)
library(ggplot2)
library(DESeq2)
library(tximport)
library(readr)
library(tximportData)
library(readxl)
library(knitr)
library(tidyverse)
library(pheatmap)
library(RColorBrewer)
library(viridis)
library(ggrepel)
library(EnhancedVolcano)
library(fgsea)
library(limma)
library(VennDiagram)
library(UpSetR)
library(wesanderson)
library(kableExtra)
library(reshape)
| Sample | patient | reads | %Q30 | Duplication rate | % reads with adapter | STAR alignment number | percent aligned | splices annotated | salmon mapping |
|---|---|---|---|---|---|---|---|---|---|
| CD123+ | HTB336 | 28161997 | 93 | 40 | 2.7 | 24795059 | 88.04 | 8802685 | 82.8170 |
| CD123+ | HTB61 | 17643949 | 90 | 29 | 2.5 | 13932164 | 78.96 | 3351481 | 68.1966 |
| CD123+ HHT | HTB336 | 17398408 | 93 | 42 | 2.7 | 15452341 | 88.81 | 5925372 | 87.9806 |
| CD123+ HHT | HTB61 | 22970807 | 93 | 33 | 2.6 | 20607839 | 89.71 | 4897461 | 75.8607 |
This table shows that there are relatively high duplication levels in these samples. This likely indicates that the input was low or too many PCR cycles were performed.
| SampleName | patient | cellType | percentrRNA |
|---|---|---|---|
| HTB61_CD123 | HTB61 | 123pos | 0.30 |
| HTB61_CD132_HHT | HTB61 | 123pos_HHT | 0.14 |
| HTB336_CD123_pos | HTB336 | 123pos | 0.38 |
| HTB336_CD123_pos_HHT | HTB336 | 123pos_HHT | 0.17 |
There was little rRNA contamination in these samples. This is expected as these are from polyA selected libraries.
There are 5001 genes with more than 5 counts in all samples
We see higher corrlation between samples than between treatments. This is expected for patient data.
We see a stronger correlation between patient than between sample type. PC1 is seperating the patients and the major loadings of this component are IFI30, HBB, MGST1, AHSP, and CREM. PC2 is separating based on treatment and the major loadings of this component are FOS, IGHM, PUF60, SLC25A6, and ICAM.
| external_gene_name | padj | pvalue | log2FoldChange |
|---|---|---|---|
| FOS | 3.44e-16 | 2.40e-20 | -4.45 |
| LRPAP1 | 4.77e-14 | 6.65e-18 | -4.62 |
| TM4SF1 | 7.62e-11 | 1.59e-14 | 4.26 |
| OSM | 3.73e-10 | 1.30e-13 | -5.03 |
| HBA2 | 3.73e-10 | 1.26e-13 | -3.61 |
| ICAM1 | 2.69e-09 | 1.13e-12 | -2.96 |
| CD83 | 6.11e-09 | 2.98e-12 | -2.88 |
| HBA1 | 1.86e-07 | 1.04e-10 | -5.37 |
| WASH5P | 5.59e-07 | 3.51e-10 | -5.12 |
| ZNF14 | 5.77e-07 | 4.02e-10 | -2.74 |
| GSDME | 1.52e-06 | 1.16e-09 | -6.11 |
| RPS15 | 3.50e-06 | 3.17e-09 | -2.74 |
| ETV3 | 3.50e-06 | 3.18e-09 | -2.40 |
| SERTAD1 | 3.71e-06 | 3.62e-09 | -3.58 |
| ZNF133 | 5.18e-06 | 5.42e-09 | -5.74 |
| ZNF256 | 1.81e-05 | 2.02e-08 | -4.74 |
| IGLC2 | 1.98e-05 | 2.62e-08 | 6.19 |
| TRAF6 | 1.98e-05 | 2.39e-08 | -2.98 |
| GADD45B | 1.98e-05 | 2.51e-08 | -3.84 |
| ATP2A1 | 2.50e-05 | 3.48e-08 | 4.94 |
| RGS1 | 3.13e-05 | 4.58e-08 | -2.61 |
| SNHG17 | 4.09e-05 | 6.27e-08 | -2.44 |
| UCP2 | 4.69e-05 | 7.53e-08 | -2.56 |
| DPH6 | 5.47e-05 | 9.15e-08 | -3.96 |
| RASGEF1B | 6.50e-05 | 1.18e-07 | -2.09 |
| SIKE1 | 6.50e-05 | 1.14e-07 | -2.65 |
| SLC25A6 | 7.98e-05 | 1.56e-07 | -2.79 |
| ZNF625-ZNF20 | 7.98e-05 | 1.52e-07 | -3.09 |
| ANXA2 | 9.48e-05 | 1.92e-07 | 2.04 |
| CHMP1B-AS1 | 9.51e-05 | 1.99e-07 | -4.70 |
| NT5DC1 | 1.11e-04 | 2.40e-07 | -3.44 |
| PDE4B | 1.16e-04 | 2.68e-07 | -2.13 |
| BSG | 1.16e-04 | 2.67e-07 | -2.67 |
| PHYH | 1.20e-04 | 2.91e-07 | -3.23 |
| ZNF442 | 1.20e-04 | 2.93e-07 | -2.67 |
| CFAP20 | 1.60e-04 | 4.02e-07 | -2.53 |
| MCTP2 | 1.68e-04 | 4.53e-07 | -2.63 |
| C19orf48 | 1.68e-04 | 4.57e-07 | -2.40 |
| PUF60 | 1.68e-04 | 4.37e-07 | -2.85 |
| GSK3B | 1.71e-04 | 4.78e-07 | 3.60 |
| NUDT3 | 1.92e-04 | 5.50e-07 | 2.51 |
| NDUFS7 | 2.47e-04 | 7.24e-07 | -3.52 |
| PRMT9 | 2.69e-04 | 8.07e-07 | -3.32 |
| ATF3 | 2.87e-04 | 8.80e-07 | -2.68 |
| ESS2 | 3.10e-04 | 9.72e-07 | -4.32 |
| NUFIP1 | 3.21e-04 | 1.03e-06 | -4.80 |
| KLF10 | 4.05e-04 | 1.33e-06 | -2.21 |
| PLAUR | 5.10e-04 | 1.71e-06 | -2.09 |
| TNFAIP3 | 5.67e-04 | 1.94e-06 | -2.40 |
| IGHM | 5.75e-04 | 2.05e-06 | -2.91 |
| MUC12 | 5.75e-04 | 2.03e-06 | -5.51 |
| NLRP3 | 6.16e-04 | 2.27e-06 | -2.49 |
| IL1RL1 | 6.16e-04 | 2.28e-06 | -3.85 |
| ABHD10 | 6.52e-04 | 2.46e-06 | -3.98 |
| ZNF140 | 6.59e-04 | 2.53e-06 | -2.21 |
| ARL4A | 6.79e-04 | 2.70e-06 | -1.94 |
| CXCR2 | 6.79e-04 | 2.67e-06 | -9.28 |
| NLE1 | 6.99e-04 | 2.83e-06 | -4.05 |
| NCKAP1 | 1.05e-03 | 4.34e-06 | 3.59 |
| PPP1R15A | 1.08e-03 | 4.51e-06 | -2.76 |
| LTBP1 | 1.13e-03 | 4.80e-06 | -7.56 |
| NANS | 1.14e-03 | 4.92e-06 | -2.19 |
| WDR43 | 1.26e-03 | 5.53e-06 | -1.99 |
| CDKN1A | 1.27e-03 | 5.68e-06 | -2.04 |
| NFU1 | 1.27e-03 | 5.76e-06 | -3.16 |
| CIDEB | 1.27e-03 | 5.90e-06 | 6.16 |
| RPL8 | 1.27e-03 | 5.94e-06 | -1.77 |
| HAUS7 | 1.30e-03 | 6.24e-06 | -3.36 |
| HEMK1 | 1.30e-03 | 6.26e-06 | -2.35 |
| FFAR3 | 1.45e-03 | 7.18e-06 | -8.78 |
| ZNF394 | 1.45e-03 | 7.10e-06 | -2.08 |
| BTBD8 | 1.53e-03 | 7.70e-06 | -8.97 |
| MTX2 | 1.54e-03 | 7.83e-06 | -4.46 |
| GRAP | 1.55e-03 | 7.98e-06 | -9.19 |
| IFFO1 | 1.72e-03 | 8.98e-06 | -8.07 |
| TNFAIP6 | 1.72e-03 | 9.11e-06 | -2.49 |
| PLA2G12A | 1.75e-03 | 9.41e-06 | -3.71 |
| CLEC4E | 1.80e-03 | 9.80e-06 | -5.24 |
| RNF138 | 1.85e-03 | 1.02e-05 | -2.14 |
| PTRHD1 | 1.85e-03 | 1.03e-05 | -3.55 |
| HNRNPDL | 1.87e-03 | 1.06e-05 | -1.87 |
| RALY | 1.92e-03 | 1.10e-05 | 3.82 |
| IVD | 1.95e-03 | 1.13e-05 | -2.50 |
| R3HCC1 | 2.18e-03 | 1.28e-05 | -3.98 |
| PMAIP1 | 2.91e-03 | 1.73e-05 | -2.40 |
| H1-4 | 2.91e-03 | 1.75e-05 | -3.24 |
| FOSB | 3.00e-03 | 1.82e-05 | -2.03 |
| WDFY2 | 3.06e-03 | 1.90e-05 | 2.33 |
| ARID4A | 3.06e-03 | 1.88e-05 | -1.90 |
| NKTR | 3.07e-03 | 1.93e-05 | -1.91 |
| EIF1 | 3.46e-03 | 2.19e-05 | -1.97 |
| ASH2L | 3.59e-03 | 2.30e-05 | -2.17 |
| USP3 | 3.71e-03 | 2.41e-05 | -2.25 |
| SNX17 | 4.02e-03 | 2.64e-05 | -2.79 |
| CYB5R4 | 4.22e-03 | 2.80e-05 | -2.17 |
| POLR2E | 4.28e-03 | 2.89e-05 | -3.21 |
| BTG2 | 4.28e-03 | 2.89e-05 | -2.40 |
| RNF144B | 4.38e-03 | 3.02e-05 | -2.40 |
| LMAN2 | 4.38e-03 | 3.00e-05 | -2.78 |
| CXCR5 | 4.38e-03 | 3.08e-05 | -9.00 |
| IRAK2 | 4.38e-03 | 3.08e-05 | -2.81 |
| H2AC19 | 4.41e-03 | 3.14e-05 | -5.21 |
| USP11 | 4.52e-03 | 3.25e-05 | -2.49 |
| GFI1B | 4.53e-03 | 3.32e-05 | -2.86 |
| ACSF3 | 4.53e-03 | 3.30e-05 | -3.43 |
| DOCK7 | 4.54e-03 | 3.40e-05 | 3.45 |
| SLC7A6OS | 4.54e-03 | 3.36e-05 | -3.84 |
| HSPA5 | 4.54e-03 | 3.42e-05 | -1.82 |
| JDP2 | 5.26e-03 | 4.00e-05 | 4.12 |
| 5.35e-03 | 4.10e-05 | -8.86 | |
| ZBTB48 | 5.53e-03 | 4.28e-05 | -2.76 |
| RGS2 | 6.13e-03 | 4.83e-05 | -2.06 |
| 6.13e-03 | 4.82e-05 | 4.33 | |
| ZNF136 | 6.19e-03 | 4.92e-05 | -1.96 |
| SAMSN1 | 6.33e-03 | 5.12e-05 | -1.74 |
| ZFP36 | 6.33e-03 | 5.16e-05 | -2.29 |
| SPPL2A | 6.33e-03 | 5.09e-05 | 2.38 |
| SRSF10 | 6.50e-03 | 5.34e-05 | -1.67 |
| POC5 | 6.79e-03 | 5.68e-05 | -3.00 |
| SNAPC1 | 6.79e-03 | 5.68e-05 | -2.08 |
| HLA-E | 7.10e-03 | 5.99e-05 | -1.62 |
| CDC37L1 | 7.17e-03 | 6.10e-05 | -2.62 |
| SDE2 | 7.40e-03 | 6.35e-05 | -1.76 |
| MBNL3 | 7.42e-03 | 6.42e-05 | -4.55 |
| DNAJB1 | 8.24e-03 | 7.18e-05 | -1.82 |
| FLRT2 | 8.45e-03 | 7.42e-05 | 3.48 |
| MIR222HG | 8.68e-03 | 7.72e-05 | 2.52 |
| CISH | 8.68e-03 | 7.75e-05 | -3.55 |
| HIF1AN | 9.24e-03 | 8.31e-05 | -2.14 |
| UGGT2 | 9.36e-03 | 8.49e-05 | -4.59 |
| DDA1 | 9.43e-03 | 8.61e-05 | -3.39 |
| LIPN | 1.00e-02 | 9.20e-05 | -8.60 |
Table showing the 132 genes with significant BH adjusted p- value of <.01.
###MA plots
This PCA plot shows the first 2 principle components following normalization with DEseq2. Again we see PC1 separating by the patient with the top gene loadings of KYNU, MTC01P12, SLAMF7, CCL7, and PPBP. The PC2 is separating on the treatment and the top gene loadings are LRPAP, FOS, TM4SF1, HBA1/2, and WASH5P.
##repeating with KEGG pathways
I can do this for any of interest, just let me know
I can do this for any of interest, just let me know
## Session Information
sessionInfo()
## R version 4.1.0 (2021-05-18)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur 10.16
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRblas.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib
##
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
##
## attached base packages:
## [1] grid parallel stats4 stats graphics grDevices utils
## [8] datasets methods base
##
## other attached packages:
## [1] reshape_0.8.8 kableExtra_1.3.4.9000
## [3] wesanderson_0.3.6 UpSetR_1.4.0
## [5] VennDiagram_1.6.20 futile.logger_1.4.3
## [7] limma_3.48.3 fgsea_1.18.0
## [9] EnhancedVolcano_1.10.0 ggrepel_0.9.1
## [11] viridis_0.6.1 viridisLite_0.4.0
## [13] RColorBrewer_1.1-2 pheatmap_1.0.12
## [15] forcats_0.5.1 stringr_1.4.0
## [17] purrr_0.3.4 tidyr_1.1.3
## [19] tibble_3.1.4 tidyverse_1.3.1
## [21] knitr_1.34 readxl_1.3.1
## [23] tximportData_1.20.0 readr_2.0.1
## [25] tximport_1.20.0 DESeq2_1.32.0
## [27] SummarizedExperiment_1.22.0 Biobase_2.52.0
## [29] MatrixGenerics_1.4.3 matrixStats_0.60.1
## [31] GenomicRanges_1.44.0 GenomeInfoDb_1.28.2
## [33] IRanges_2.26.0 S4Vectors_0.30.0
## [35] BiocGenerics_0.38.0 ggplot2_3.3.5
## [37] dplyr_1.0.7
##
## loaded via a namespace (and not attached):
## [1] backports_1.2.1 fastmatch_1.1-3 BiocFileCache_2.0.0
## [4] systemfonts_1.0.2 plyr_1.8.6 splines_4.1.0
## [7] crosstalk_1.1.1 BiocParallel_1.26.2 digest_0.6.27
## [10] htmltools_0.5.2 fansi_0.5.0 magrittr_2.0.1
## [13] memoise_2.0.0 tzdb_0.1.2 Biostrings_2.60.2
## [16] annotate_1.70.0 modelr_0.1.8 extrafont_0.17
## [19] vroom_1.5.4 extrafontdb_1.0 svglite_2.0.0
## [22] prettyunits_1.1.1 colorspace_2.0-2 rappdirs_0.3.3
## [25] blob_1.2.2 rvest_1.0.1 haven_2.4.3
## [28] xfun_0.26 crayon_1.4.1 RCurl_1.98-1.4
## [31] jsonlite_1.7.2 genefilter_1.74.0 survival_3.2-13
## [34] glue_1.4.2 gtable_0.3.0 zlibbioc_1.38.0
## [37] XVector_0.32.0 webshot_0.5.2 DelayedArray_0.18.0
## [40] proj4_1.0-10.1 Rttf2pt1_1.3.9 maps_3.3.0
## [43] scales_1.1.1 futile.options_1.0.1 DBI_1.1.1
## [46] Rcpp_1.0.7 progress_1.2.2 xtable_1.8-4
## [49] bit_4.0.4 DT_0.18 htmlwidgets_1.5.3
## [52] httr_1.4.2 ellipsis_0.3.2 farver_2.1.0
## [55] pkgconfig_2.0.3 XML_3.99-0.7 sass_0.4.0
## [58] dbplyr_2.1.1 locfit_1.5-9.4 utf8_1.2.2
## [61] labeling_0.4.2 tidyselect_1.1.1 rlang_0.4.11
## [64] AnnotationDbi_1.54.1 munsell_0.5.0 cellranger_1.1.0
## [67] tools_4.1.0 cachem_1.0.6 cli_3.0.1
## [70] generics_0.1.0 RSQLite_2.2.8 broom_0.7.9
## [73] evaluate_0.14 fastmap_1.1.0 yaml_2.2.1
## [76] bit64_4.0.5 fs_1.5.0 KEGGREST_1.32.0
## [79] ash_1.0-15 formatR_1.11 ggrastr_0.2.3
## [82] xml2_1.3.2 biomaRt_2.48.3 compiler_4.1.0
## [85] rstudioapi_0.13 filelock_1.0.2 curl_4.3.2
## [88] beeswarm_0.4.0 png_0.1-7 reprex_2.0.1
## [91] geneplotter_1.70.0 bslib_0.2.5.1 stringi_1.7.4
## [94] highr_0.9 ggalt_0.4.0 lattice_0.20-44
## [97] Matrix_1.3-4 vctrs_0.3.8 pillar_1.6.2
## [100] lifecycle_1.0.0 jquerylib_0.1.4 data.table_1.14.0
## [103] bitops_1.0-7 R6_2.5.1 KernSmooth_2.23-20
## [106] gridExtra_2.3 vipor_0.4.5 lambda.r_1.2.4
## [109] MASS_7.3-54 assertthat_0.2.1 withr_2.4.2
## [112] GenomeInfoDbData_1.2.6 hms_1.1.0 rmarkdown_2.11
## [115] lubridate_1.7.10 ggbeeswarm_0.6.0